Co-Treatment with Milatuzumab (Anti-CD74 mAb) and Rituximab (Anti-CD20 mAb) Results in the Induction of Mantle Cell Lymphoma Cell Death That Is Dependent On Actin Polymerization and Inhibition of NF-Kb.

Abstract Abstract 1694 Poster Board I-720 Mantle cell lymphoma (MCL) is an incurable B-cell malignancy and patients with this disease have limited therapeutic options. Despite the success of rituximab in treatment of B-cell lymphoma, its use as a single agent or in combination with chemotherapy in MCL has demonstrated modest activity; thus, novel strategies are needed. CD74 is an integral membrane protein expressed on malignant B cells and is implicated in promoting survival and growth, making it an attractive therapeutic target. The humanized anti-CD74 monoclonal antibody (mAb), milatuzumab, (Immunomedics) has shown promising preclinical activity against several human B-cell lymphoma cell lines, but has not been studied in MCL. Since rituximab and milatuzumab target distinct antigens lacking known association, we explored a combination strategy with these mAbs in MCL cell lines, patient samples, and in a preclinical model of MCL. Flow cytometric analysis shows that 6 different MCL cell lines (Mino, JeKo, SP53, Rec-1, Hbl2, Granta-519) and MCL patient primary tumor cells, express variable levels of CD74, with Mino cells showing highest level and Rec-1 the lowest. Incubation of the 6 MCL cell lines and primary cells (7 patients) with immobilized milatuzumab (5 μg/ml) and rituximab (10 μg/ml) resulted in mitochondrial depolarization and in statistically significant enhanced induction of apoptosis determined by Annexin V/PI and flow cytometry. The combination of both agents resulted in additive induction of apoptosis that was caspase independent in 5 MCL cell lines (synergistic in JeKo cells) and in primary cells, at 8, 24 and 48 hours. Importantly, while sensitivity to milatuzumab depends on the level of CD74 expression, the combination of milatuzumab and rituximab was able to induce enhanced cell death in all MCL cell lines and MCL primary cells, regardless of antigen density. We demonstrated that the combination of milatuzumab and rituximab induced enhanced apoptosis in a caspase-independent fashion with no apparent involvement of apoptotic key regulatory proteins such as Bax, Bcl-2, Bcl-Xl and Mcl-1. However, changes in the nuclear level of p65 were observed with either drug alone and with the combination, starting as early as 4 hours after treatment. The association of CD74 with MHC class II led us to explore pro-death mechanisms that become operable during HLA-DR-specific mAb treatment of lymphoma cells (Ivanov A et al., J Clin Invest 2009). We therefore investigated the role of actin polymerization by addition of cytochalasin D and latrunculin B, inhibitors of actin polimerization, prior to treatment with milatuzumab and/or rituximab. These studies showed that milatuzumab-induced MCL cell (Jeko and Mino) death was dependent on actin polymerization. To examine the in vivo activity of rituximab and milatuzumab, a preclinical model of human MCL using the SCID (CB17 scid/scid) mouse depleted of NK cells with TMβ1 mAb (anti-murine IL2Rb) was used. In this model, i.v. injection of 40×106 JeKo cells results in disseminated MCL 3 weeks after engraftment. The primary end-point was survival, defined as the time to develop cachexia/wasting syndrome or hind limb paralysis. Ten mice/group were treated starting at day 15 post-engraftment with intraperitoneal trastuzumab mAb control (300 μg qod), milatuzumab (300 μg qod), rituximab (300 μg qod), or a combination of milatuzumab and rituximab. The mean survival for the combination-treated group was 44.5 days (95%CI:39,51), compared to 28 days for trastuzumab-treated mice (95% CI:24,30), 33.5 days for the milatuzumab-treated mice (95% CI:28,36), and 38 days for the rituximab-treated mice (95%CI:36,42). The combination treatment prolonged survival of this group compared to trastuzumab control (P<0.0001), milatuzumab (P<0.0001) or rituximab (P=0.03). No overt toxicity from milatuzumab or the combination regimen was noted. These preliminary results provide justification for further evaluation of milatuzumab and rituximab in combination in MCL. Disclosures Off Label Use: Milatuzumab for Mantle Cell Lymphoma Treatment. Goldenberg:Immunomedics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

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Combination Anti-CD74 (Milatuzumab) and CD20 (Rituximab) Monoclonal Antibody Therapy Has in Vitro and in Vivo Activity in Mantle Cell Lymphoma

Blood ◽

10.1182/blood.v112.11.886.886 ◽

2008 ◽

Vol 112 (11) ◽

pp. 886-886 ◽

Cited By ~ 1

Author(s):

Lapo Alinari ◽

Erin Hertlein ◽

David M. Goldenberg ◽

Rosa Lapalombella ◽

Fengting Yan ◽

...

Keyword(s):

Monoclonal Antibody ◽

B Cell ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Combination Treatment ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Preclinical Model ◽

Mantle Cell

Abstract Mantle cell lymphoma (MCL) is an incurable B-cell malignancy and patients with this disease have limited therapeutic options. Despite the success of Rituximab in treatment of B-cell malignancies, its use as a single agent or in combination with chemotherapy in MCL has demonstrated modest activity; thus, novel strategies are needed. CD74 is an integral membrane protein expressed on malignant B cells and implicated in promoting survival and growth, making it an attractive therapeutic target. The humanized anti-CD74 monoclonal antibody (mAb), Milatuzumab, (Immunomedics) has shown promising preclinical activity against several human B-cell lymphoma cell lines, but has not been studied in MCL. Since Rituximab and Milatuzumab target distinct antigens lacking known association, we explored a combination strategy with these mAbs in MCL cell lines, patient samples, and in a preclinical model of MCL. Flow cytometric analysis shows that the MCL cell lines Mino and JeKo, and MCL patient tumor cells, express abundant surface CD74 compared to the CD74-negative cell line, Jurkat. Incubation of Mino and JeKo cells with immobilized (goat anti-human IgG) Milatuzumab (5 μg/ml) resulted in mitochondrial depolarization and significant induction of apoptosis determined by Annexin V/PI and flow cytometry (apoptosis at 8hr=38.3±0.85% and 25.4±2.6%; 24hr=73.6±3.47% and 36±3.57%; 48hr=84.9±3.91% and 50.4±4.17%, respectively, compared to Trastuzumab (control). Expression of surviving cells from anti-CD74-treated MCL cells consistently demonstrated marked induction of surface CD74 (MFI 762) compared to control (MFI 6.1). Incubation with immobilized Rituximab (10 μg/ml) resulted in 39.5±2.5% and 37.1±8.35% apoptotic events at 8hr, 58.8±3.14%, 41.2±8.27% at 24hr, and 40.1±1.3% and 45.6±3.25% at 48hr, respectively. Combination treatment of Mino and JeKo cells with Milatuzumab and Rituximab led to significant enhancement in cell death, with 77.6±3.95% and 79.6±2.62% apoptosis at 8hr in Jeko and Mino cells (P=0.0008 and P=0.00004 vs. Milatuzumab alone; P=0.00015 and P=0.001 vs. Rituximab alone); 90.4±3.53% and 76.6±4.3% at 24hr, respectively (P=0.0042 and P=0.0002 vs. Milatuzumab, P=0.0003 and P=0.0027 vs. Rituximab alone); 92.8±0.77% and 85.6±2.62% at 48hr, respectively (P= 0.026 and P=0.0002 vs. Milatuzumab alone, P=0.0000005 and P=0.00008 compared to Rituximab alone, respectively). To examine the in vivo activity of Rituximab and Milatuzumab, a preclinical model of human MCL using the SCID (cb17 scid/scid) mouse depleted of NK cells with TMβ1 mAb (anti-murine IL2Rb) was used. In this model, intravenous injection of 40×106 JeKo cells results in disseminated MCL 3–4 weeks after engraftment. The primary end-point was survival, defined as the time to develop cachexia/wasting syndrome or hind limb paralysis. Mice were treated starting at day 17 postengraftment with intraperitoneal Trastuzumab mAb control (300 μg qod), Milatuzumab (300 μg qod), Rituximab (300 μg qod), or a combination of Milatuzumab and Rituximab. The mean survival for the combination-treated group was 55 days (95%CI:41, upper limit not reached as study was terminated at day 70), compared to 33 days for Trastuzumab-treated mice (95% CI:31,34), 35.5 days for the Milatuzumab-treated mice (95% CI:33,37), and 45 days for the Rituximab-treated mice (95%CI:30,46). The combination treatment prolonged survival of this group compared to Trastuzumab control (P=0.001), Milatuzumab (P=0.0006) and Rituximab (P=0.098). No overt toxicity from Milatuzumab or the combination regimen was noted. A confirmatory study with a larger group of mice and detailed mechanistic studies are now underway. These preliminary results provide justification for further evaluation of Milatuzumab and Rituximab in combination in MCL.

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Mantle-Cell Lymphoma (MCL) Cells Are Highly Sensitive To ABT-199 But Their Sensitivity May Be Altered By The Microenvironment Via The Up-Regulation Of Bcl-Xl and Bcl2A1

Blood ◽

10.1182/blood.v122.21.4285.4285 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4285-4285 ◽

Cited By ~ 1

Author(s):

Cyrille Touzeau ◽

Carole Brosseau ◽

Christelle Dousset ◽

Catherine Pellat-Deceunynck ◽

Steven Le Gouill ◽

...

Keyword(s):

Antitumor Activity ◽

Lymph Nodes ◽

B Cell ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Primary Cells ◽

Highly Sensitive ◽

Mantle Cell

Abstract Despite improvement in the treatment of Mantle-cell lymphoma (MCL), relapse invariably occurs and innovative strategies are needed. Bcl-2 inhibitors such as ABT-737 and ABT-263 (navitoclax), which target both Bcl-2 and Bcl-xL, demonstrated antitumor activity in B-cell malignancies. However, the clinical development of navitoclax is limited by a deep thrombocytopenia, which is induced by inhibition of Bcl-xL in platelets. To overcome this toxicity, ABT-199, the first-in-class orally bioavailable Bcl-2-selective BH3 mimetic, has been developed and showed promising antitumor activity in B-cell lymphoma while sparing platelets. In the present study, the apoptotic efficiency of ABT-199 in comparison with that of ABT-737 was evaluated in seven MCL cell lines. We found two MCL cell lines sensitive to ABT-199 (LD50 of 100 and 200 nM), one intermediate (LD50 of 1000nM) and 4 resistant (LD50 from 5000 to 10000 nM). Surprisingly, LD50 values of the 2 sensitive cell lines (MINO, GRANTA-519) were slightly higher for ABT-199 than for ABT-737. We further demonstrated that the Bcl-2/Mcl-1 ratio determined by RT-PCR is a predictive biomarker for ABT-199 sensitivity. To further determine the role of Mcl-1 in ABT-199 resistance, Mcl-1 siRNA were transfected in Z-138 and JEKO-1 cells. Mcl-1 silencing sensitized these 2 cell lines to low dose ABT-199 confirming the importance of Mcl-1 in ABT-199 resistance as previously shown for ABT-737. Moreover, in Z-138 cells, which highly express Bcl-xL, we showed that Bcl-xL silencing sensitized them to ABT-199. These results show that in addition to Mcl-1, Bcl-xL might also confer resistance to ABT-199-induced apoptosis in MCL. This could explain the slight difference of sensitivity of MCL cells between ABT-199 and ABT-737. In contrast to MCL cell lines, we found so far that ABT-199 efficiency killed all tested circulating primary cells from MCL patients (n=7) with LD50 values inferior to 10 nM. Because MCL cells reside mainly in lymph nodes, we wondered whether mimicking the microenvironment could impact the sensitivity of MCL cells to BH3 mimetics like it was previously demonstrated for chronic lymphoid leukemia cells. Thus, the ABT-199 sensitive MINO and GRANTA-519 cells were cultured on CD40L-expressing fibroblasts L in order to mimic the lymph node microenvironment. Both cell lines and primary cells became resistant to ABT-199 within 24h. Investigation of the underlying mechanism revealed a strong up-regulation of both Bcl-xL and Bcl2A1 protein expression. By contrast, culture of MCL cells with parental CD40L- fibroblasts or in conditioned medium from CD40L+ L fibroblasts culture failed to induce ABT-199 resistance. These results highlight the implication of the CD40L pathway in ABT-199 resistance through the up-regulation of Bcl-xL and Bcl2A1 in MCL. In conclusion, while circulating primary MCL cells are highly sensitive to ABT-199, it would be important to address the impact of microenvironment on long-term survival of MCL cells within lymph nodes under ABT-199 treatment. Disclosures: No relevant conflicts of interest to declare.

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Forodesine (Immucillin H, BCX-1777) Shows Activity on Mantle Cell Lymphoma Primary Cells

Blood ◽

10.1182/blood.v112.11.4997.4997 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4997-4997

Author(s):

Andrea Rinaldi ◽

Emilia Ceresa ◽

Davide Rossi ◽

Gianluca Gaidano ◽

Shanta Bantia ◽

...

Keyword(s):

B Cell ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Cell Lymphoma ◽

Annexin V ◽

B Cell Lymphoma ◽

B Cell Malignancies ◽

New Therapeutic Approaches ◽

Mantle Cell

Abstract Mantle cell lymphoma (MCL) represents a subtype of B-cell lymphoma associated with a very unfavourable clinical outcome. Currently no therapy can be considered as standard, and new therapeutic approaches are needed. Forodesine is a potent inhibitor of purine nucleoside phosphorylase (PNP), whose major role is to catalyze the cleavage of inosine, deoxyinosine guanosine, and deoxyguanosine (dGuo) to their corresponding base and sugar 1-phosphate by phosphorolysis. In the presence of deoxycytidine kinase, PNP inhibition leads to an increase in the concentration of dGuo triphosphate (dGTP), followed by inhibition of DNA synthesis and cell death by apoptosis. When combined with dGuo, forodesine has been shown to have in vitro cytotoxic activity on T-cell (T-ALL, T-PLL) and on B-cell malignancies (CLL, B-ALL), and Phase I/II trials are on going in CLL and CTCL patients. Here, we report the first data on in vitro activity of forodesine in MCL. Primary MCL cells, derived from six patients, were exposed to forodesine (0, 2, 20 μM) in combination with dGuo (0, 10, 20 μM), for 48 hrs. Cells were cultured in X-VIVO 10 medium (Cambrex) with 10% FBS. Cell viability was assessed by flow cytometry with the Annexin V - propidium iodide assay. Four patient samples (67%) showed an increase in the number of Annexin V positive cells ranging from 1.9 to 5.3 times compared to untreated cells. The effect was larger for 20 μM forodesine compared with 2 μM. There was no effect of dGuo alone and only a minimal effect of increasing dGuo concentration from 10 μM to 20 μM. Cell lines did not appear to be ideal models to evaluate the efficacy of forodesine in vitro. Three established MCL cell lines (Granta-519, Rec, JeKo1) were treated with escalating doses of forodesine, but the results were not reproducible, while the same cells showed expected IC50 values between 25–30 μM when exposed to bendamustine for 72 hrs. In conclusion, the in vitro data reported here with 4/6 MCL patients primary samples sensitive to forodesine and the results from various groups on other T- and B-cell malignancies suggest that clinical trials of forodesine in MCL may be warranted.

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Targeting Transcription Checkpoint Using a Novel CDK9 Inhibitor in Mantle Cell Lymphoma

Blood ◽

10.1182/blood-2020-140865 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 28-29

Author(s):

Junwei Lian ◽

Yu Xue ◽

Alexa A Jordan ◽

Joseph McIntosh ◽

Yang Liu ◽

...

Keyword(s):

B Cell ◽

Mantle Cell Lymphoma ◽

Mouse Models ◽

Cell Lines ◽

Cell Apoptosis ◽

Research Funding ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Mantle Cell ◽

Cdk9 Inhibitor

Introduction Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma that accounts for 5-8% of all non-Hodgkin lymphomas. Despite the Bruton's tyrosine kinase inhibitor ibrutinib and the BH3 mimetic BCL2 inhibitor venetoclax (ABT-199) have proven to be effective therapeutic strategies for MCL, most patients often experience disease progression after treatment. Thus, developing a novel drug to overcome this aggressive relapsed/refractory malignancy is an urgent need. Cyclin-dependent kinase 9 (CDK9) is a serine/threonine kinase belonging to the CDK family which regulates multiple cellular processes, particularly in driving and maintaining cancer cell growth. Unlike classical CDKs, CDK9 is a critical component of the positive transcription elongation factor b (P-TEFb) complex that mediates transcription elongation and mRNA maturation via phosphorylating RNA polymerase II (RNAP2). Previous studies demonstrated that CDK9 inhibition downregulates transcription levels of MCL-1 and MYC, which are crucial in both survival and proliferation of acute myeloid leukemia and diffuse large B-cell lymphoma. We and others found that the MYC signaling pathway was enhanced in MCL, especially in ibrutinib-resistant MCL patients. MYC is a core transcription factor driving lymphomagenesis. It does not possess enzymatic activity and has long been considered to be undruggable. MCL-1 is a key anti-apoptotic protein and is overexpressed in several hematologic malignancies. It was also found to be overexpressed in ibrutinib or venetoclax-resistant MCL cells. Thus, CDK9 is considered as a potential target that may inhibit MYC and MCL-1 pathways. Although recently it was shown that MC180295, a novel selective inhibitor of CDK9, has nanomolar levels anti-cancer potency, whether its beneficial effects extend to relapsed/refractory MCL has not yet been assessed. Methods We use three paired MCL cells sensitive/resistant to ibrutinib or venetoclax to test the efficacy of CDK9 inhibitor MC180295. Cell viability was measured by using Cell Titer Glo (Promega). Cell apoptosis assay and western blot analyses were used to identify affected pathways after MC180295 treatment. Finally, we used patient-derived xenograft (PDX) mouse models to test the therapeutic potential of MC180295 in MCL. Results First, we examined the potential efficacy of a CDK9 inhibitor MC180295 in MCL cells. MC180295 treatment results in growth inhibition of ibrutinib-resistant or venetoclax-resistant MCL cells. By assessing the caspase 3 and PARP activity, we found that MC180295 treatment induces cell death via cell apoptosis in MCL cell lines. Meanwhile, we found that RNAP2 phosphorylation at Ser2, the active form of RNAP2, is downregulated in MC180295 treated MCL cell lines. Consistent to previous studies, MC180295 treatment significantly reduces the protein level of MYC and MCL-1. In addition, we identified several other important proteins, such as cyclin D1 and BCL-XL, were also downregulated upon MCL180295 treatment. MC180295 was able to overcome ibrutinib-venetoclax dual resistance in PDX mouse models without severe side effects. To improve the efficacy of MC180295 as a single agent, we performed in vitro combinational drug screen with a number of FDA-approved or investigational clinical agents and found that MC180295 had a synergistic effect with venetoclax. We are currently investigating the underlying mechanism of action. Conclusion Taken together, our findings showed that targeting CDK9 by its specific inhibitor MC180295 is effective in targeting MCL cells, especially those with ibrutinib or venetoclax resistance and therefore supports the concept that CDK9 is a new target to overcome ibrutinib/venetoclax resistance in MCL. Disclosures Wang: MoreHealth: Consultancy; Dava Oncology: Honoraria; Beijing Medical Award Foundation: Honoraria; OncLive: Honoraria; Molecular Templates: Research Funding; Verastem: Research Funding; Guidepoint Global: Consultancy; Nobel Insights: Consultancy; Oncternal: Consultancy, Research Funding; InnoCare: Consultancy; Loxo Oncology: Consultancy, Research Funding; Targeted Oncology: Honoraria; OMI: Honoraria, Other: Travel, accommodation, expenses; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Lu Daopei Medical Group: Honoraria; Pulse Biosciences: Consultancy; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Juno: Consultancy, Research Funding; BioInvent: Research Funding; VelosBio: Research Funding; Acerta Pharma: Research Funding.

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Targeting the PI3K/AKT/Survivin Pathway with Ritonavir In Therapy-Resistant Mantle Cell Lymphoma

Blood ◽

10.1182/blood.v116.21.2855.2855 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2855-2855

Author(s):

Tara M Nordgren, ◽

Ganapati Hegde ◽

Corey Munger ◽

Julie M. Vose ◽

Shantaram Joshi

Keyword(s):

B Cell ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Cell Lymphoma ◽

Combined Treatment ◽

Flow Cytometric Analysis ◽

B Cell Lymphoma ◽

Qrt Pcr ◽

Mantle Cell

Abstract Abstract 2855 Mantle cell lymphoma (MCL) is an aggressive B cell lymphoma accounting for about 6% of non-Hodgkin's lymphoma cases in the US. While multiple therapy regimens are available to treat MCL, patients ultimately relapse within 3–4 years from therapy-resistant MCL, making MCL carry the worst prognosis of all non-Hodgkin's B cell lymphomas. To improve therapies for patients with MCL, we must understand the biological causes for relapse and therapy-resistance in MCL and develop mechanisms to target the unique properties in relapsing MCL. Recently, we have isolated and produced therapy-resistant MCL cell lines by inoculating NOD-SCID mice with a human MCL cell line, Granta-519 (GP), and subsequently treating with CHOP chemotherapy regimen plus bortezomib. When mice relapsed following therapy, tumor cells were isolated from the kidneys and livers, and cultured to produce stable cell lines, named GRK and GRL, respectively. GRK and GRL cells exhibited increased proliferative and therapy-resistant properties in vitro, and GRL showed increased aggressiveness in vivo also, as compared with GP. Using quantitative PCR (qRT-PCR) to assess the activation of pathways relating to lymphoma progression, the PI3K/Akt/survivin pathway was found to be differentially activated in GRL and GRK compared to GP, including a 6.5 fold increase in survivin in GRL. Therefore, we inhibited survivin in GRL using the FDA-approved protease inhibitor ritonavir (Abbott Laboratories), as recent studies suggest this compound is able to downregulate survivin in lymphoblastoid B cells (Dewan, et al. Int J Cancer 2009; 124[3]: 622–629). When GRL cells were incubated with ritonavir plus vincristine or doxorubicin, MTT assays showed significant inhibition of cell proliferation/survival (p < 0.05) compared to the effects of ritonavir alone. Also, flow cytometric analysis of Annexin V demonstrated dose-dependent induction of apoptosis upon ritonavir treatment in GRL. In addition, qRT-PCR analysis demonstrated a decrease in mRNA expression of the pro-survival genes cyclin D2, Rel A, survivin, and BCL2 in GP and GRL cells after ritonavir treatment. Together, these studies demonstrate the potentials of utilizing ritonavir in a combined treatment regimen designed to target therapy-resistant MCL. As ritonavir is already an FDA-approved drug, these studies hold promise for expediting future in vivo and clinical studies that may eliminate therapy-resistant cells responsible for relapse in MCL patients.(This research was supported by the Lymphoma Research Foundation, New York, NY). Disclosures: No relevant conflicts of interest to declare.

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In Vitro Combination of Bortezomib with Enzastaurin or Lenalidomide Enhances the Cytotoxicity in B-Cell Lymphoma Cell Lines.

Blood ◽

10.1182/blood.v120.21.2754.2754 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2754-2754 ◽

Cited By ~ 1

Author(s):

Maria Cosenza ◽

Monica Civallero ◽

Samantha Pozzi ◽

Alessia Bari ◽

Eliana Valentina Liardo ◽

...

Keyword(s):

Bone Marrow ◽

Cell Proliferation ◽

Follicular Lymphoma ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Primary Cells ◽

Mantle Cell

Abstract Abstract 2754 Background. Therapy for patients with non-Hodgkin's Lymphomas (NHL) have significantly improved over the last decade, especially since the discovery of monoclonal antibodies and other biologic therapies. Although patients with B-cell NHL usually respond to conventional chemotherapy, they often relapse in spite of salvage therapy and stem cell transplantation. Early clinical studies of Bortezomib-based combinations, showed encouraging results both in Follicular Lymphoma (FL) as well as in Mantle Cell Lymphomas (MCL). In this study we hypothesize that combining Bortezomib with Enzastaurin or Lenalidomide would target separate signaling pathways increasing tumor-cell death. Methods. Bortezomib, Lenalidomide and Enzastaurin alone and their combinations were tested in WSU-NHL, RL (FL cell lines) and Granta-519 and Jeko-1 (MCL cell lines) and primary cells from lymphoma patients. B-NHL cell lines were treated for 24–48 hours. The cell proliferation was determined by using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit and cell cytotoxicity with MTT-assay. The interaction between drugs was evaluated by isobologram analysis using the STACorp 8.2 software program based upon the Chou-Talalay method to determine if the combination were additive or synergistic. Apoptosis was evaluated by flow cytometry using Annexin V/Propidium Iodide (PI) staining. The effect on cell cycle was analyzed using PI by flow cytometry. Western blotting experiments were performed to determine whether the drugs combinations affected PI3K/Akt, PKC and MAPK/ERK pathways. Results. In the present study we have shown that Enzastaurin and Lenalidomide enhanced the cytotoxicity of Bortezomib in all B-NHL cell lines and primary cells from lymphoma patients. A clear synergistic interaction, confirmed by the Chou-Talalay method (combination index<1) was observed after 24 hours using low concentrations of all the drugs (Bortezomib 6 nM + Lenalidomide 6 μM; Bortezomib 6 nM + Enzastaurin 6 μM). The combination of Bortezomib with both Enzastaurin or Lenalidomide did not trigger relevant decrease in the viability of normal peripheral blood mononuclear cells (PBMNCs) and suppressed cell proliferation of B-NHL cell lines when co-cultured with bone marrow stromal cells (BMSCs) in a system that mimics the bone marrow microenvironment. In comparison with each single agents, the combination of Bortezomib with both Enzastaurin and Lenalidomide induced significant increase of apoptosis. This enhancement of apoptosis is mediated by an increased ratio of pro-apoptotic protein (Bim, Bad) to anti-apoptotic proteins (Bcl-2, Bcl-xL) which increased the threshold for caspases 3 and 9. The cycle analysis showed that the combination of Bortezomib with both Enzastaurin or Lenalidomide reduced the proportion of cells in the G0/G1, S and G2/M phase, increasing sub G0/G1. Western blot analysis showed that anti-proliferative events and pro-apoptotic effects were associated with dephosphorylation of PI3K/Akt and MAPK/ERK pathways. Conclusion. In this study, we investigated the direct antitumor activity of Bortezomib combined with Enzastaurin or Lenalidomide in established B-NHL cells (Follicular Lymphoma and Mantle Cell Lymphoma) and freshly isolated patients cells in vitro. Our results demonstrated that the combination of Bortezomib with both Enzastaurin and Lenalidomide induces synergistic anti-proliferative and pro-apoptotic effects in all B-cell lymphoma cell lines and primary cells, even in the presence of the bone marrow microenvironment. This direct cytoxicity is mediated by signaling events involving PI3K/Akt, MAPK/ERK and Bcl-2 pathways leading to cell death. Hence, this in vitro studies to test combinations of these active agents in patients with Follicular Lymphoma and Mantle Cell Lymphoma. Disclosures: No relevant conflicts of interest to declare.

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Prospective clinical study of R-CMD therapy for indolent B cell lymphoma and mantle cell lymphoma from the Hokuriku Hematology Oncology Study Group

Medical Oncology ◽

10.1007/s12032-015-0677-9 ◽

2015 ◽

Vol 32 (9) ◽

Author(s):

Tomoyuki Sakai ◽

Yasufumi Masaki ◽

Nozomi Otsuki ◽

Ippei Sakamaki ◽

Shinji Kishi ◽

...

Keyword(s):

Clinical Study ◽

B Cell ◽

Mantle Cell Lymphoma ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Prospective Clinical Study ◽

Study Group ◽

Mantle Cell

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Mantle Cell Lymphoma

Archives of Pathology & Laboratory Medicine ◽

10.5858/1999-123-1182-mcl ◽

1999 ◽

Vol 123 (12) ◽

pp. 1182-1188 ◽

Cited By ~ 1

Author(s):

Rebecca C. Hankin ◽

Susan V. Hunter

Keyword(s):

Polymerase Chain Reaction ◽

B Cell ◽

Mantle Cell Lymphoma ◽

Diagnostic Tests ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Molecular Diagnostic ◽

Chain Reaction ◽

Mantle Cell ◽

Polymerase Chain

Abstract Objective.—This article summarizes the most useful ancillary immunohistochemical and molecular assays for use in the diagnosis of mantle cell lymphoma. Data Sources.—The English language literature was surveyed, with an emphasis on recent publications, for articles presenting key advances in the molecular characterization of mantle cell lymphomas and for series of cases testing the utility of molecular diagnostic tests. The authors' series of 26 small B-cell lymphomas, analyzed for the cyclin D1 protein by paraffin immunohistochemistry and for t(11;14) by polymerase chain reaction, is included. Conclusions.—Mantle cell lymphoma, a B-cell lymphoma now recognized in the 1994 Revised European-American Classification of Lymphoid Neoplasms (REAL) classification, is a relatively aggressive lymphoma with a poor prognosis. Its characteristic t(11;14)(q13;q32) translocation has a role in oncogenesis and has been exploited for molecular diagnostic tests, but these tests vary in sensitivity, specificity, and ease of use. Improved immunohistochemical tests are sufficient to confirm the diagnosis in most cases. Conventional cytogenetics and molecular diagnostic tests for t(11;14)—Southern blot and polymerase chain reaction analysis—may be helpful in selected cases, but are laborious or of limited sensitivity. Other methods, such as fluorescence in situ hybridization, need further development to provide faster, more sensitive diagnosis.

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